Yogurt Benefits Bone Mineralization in Ovariectomized Rats with Concomitant Modulation of the Gut Microbiome

Scope Evidence supports that gut‐modulating foods potentially can suppress bone loss in postmenopausal women. This study aims to investigate the effect of milk calcium‐enriched milk, yogurt, and yogurt‐inulin combination on the gut–bone association. Methods and results A 6‐week intervention study is conducted in ovariectomized rats. Four pastes containing milk calcium‐fortified milk (M‐Ca), milk calcium‐fortified yogurt (Y‐Ca), inulin‐fortified Y‐Ca (Y‐I‐Ca), or an isoconcentration of calcium carbonate (Ca‐N), and a calcium‐deficient paste are provided. M‐Ca does not influence bone mineral density and content (BMD and BMC), femur mechanical strength, or femoral microstructure compared to Ca‐N, but Y‐Ca increases spine BMD. The serum metabolome reveals that Y‐Ca modulated glycine‐related pathways with reduced glycine, serine, and threonine. No additive effects of yogurt and inulin are found on bone parameters. Correlation analysis shows that increased lactobacilli and reduced Clostridiaceae members in Y‐Ca is associated with an increased spine BMD. Increases in Bifidobacterium pseudolongum, Turicibacter, Blautia, and Allobaculum and gut short‐chain fatty acids in Y‐I‐Ca are not reflected in bone parameters. Conclusion Yogurt as calcium vehicle contributes to increased spine BMD concomitant with changes in the gut microbiome and glycine‐related pathways, while adding inulin to yogurt does not affect bone mineralization in ovariectomized rats.

The yoghurt base was produced from fresh skimmed milk, which was homogenized (200/50 bar) and pasteurized (72 ºC, 15 s) prior to fermentation with culture YC-X11 (Chr. Hansen A/S, Denmark) at 42 ºC until pH 4.5 was reached. The product was cooled to 5 ºC and stored at 4 ºC until production of food paste the next day. The food pastes for the five different groups were produced by mixing the dry ingredients (Altromin powdered diet and Capolac ® MM-0525 BG) with the corresponding wet component (water, milk or yoghurt), see Table S1.
The dry ingredients for the recipes containing milk mineral (Capolac ® ) were mixed using an Inversia Tumbler Mixer equipped with a 20 liter drum (25 rpm for 25 min) to ensure a homogeneous distribution of the component in the powder prior to mixing the food paste. The diets without milk mineral were prepared from dry ingredients as delivered by Altromin.
For each recipe, the wet component was added to a Stephan Cooker UM/SK 44 equipped with a sharp knife mixing system. The dry ingredient mix was added and the paste was mixed for 2 min at 100% speed and 3000 rpm. The product was checked and mixing was continued for 5 min at 50% speed.
After mixing, 150 ± 1 g paste was transferred to 140 mL aluminum beakers with snap-on PET lids and stored at -18 ºC until served. Microbiological evaluations of the frozen products were conducted prior to release from Arla Foods Ingredients.

Method S2. X-ray micro-computed tomography
The femora were immersed in 96% EtOH and wrapped in cotton wool to keep them stable during measurement. 7700-µm high volumes of interest (VOI) containing the distal metaphysis were measured. The instrument was operated at 60 kV and 6 W with the LE2 filter inserted for attenuation of low-energy X-rays. 1601 projections were recorded over a 360° sample rotation with 2 s exposure/projection and using the 0.4× objective and a detector binning of 2 to result in an isotropic voxel size of 7.6 µm. The reconstructed tomograms were processed with Dragonfly Version 2021.3 (Object Research Systems Inc, Montreal, Canada).
2200-µm high VOIs for analysis of trabecular bone were selected starting 1500 µm above the most distal part of the growth plate following the procedure described in reference (18) .
Segmentation of trabecular bone was done by manually drawing along the endocortical surface of the cortex in every 30th image along the bone long axis followed by automatic interpolation and segmentation of marrow versus trabecular bone by Otsu's method. For analysis of cortical bone, VOIs starting from the most proximal part of the measured volume and extending 980 µm towards the distal end were segmented by Otsu's method to separate bone from marrow and surrounding EtOH. Trabecular bone was subtracted by the same semimanual procedure described above. Trabecular and cortical bone micro-structural parameters were computed for each of the two segmented VOIs using Dragonfly's Bone Analysis Wizard.

Method S3. Preparation of intestinal contents, feces and serum for NMR spectroscop
Approx. 100 mg intestinal content or feces were mixed with 200 µL Milli-Q water in a 1.5   Figure S1. Body weight variations of OVX rats fed with five different diets during a 6-week intervention. Figure S2. The correlations between bone parameters and body weight. The symbol circle represents significant correlations (p<0.05) between parameters, and the corresponding color shows the value of correlation coefficients.          Figure S15. A food paste left over after 24 hours provided to rats